Apparatus and method for culturing micro-organisms



7, 1963 J. H. BREWER 3,102,082

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United States Patent 3,102,082 APPARATUS AND METHOD FOR CULTURINGMICRO-ORGANISMS John H. Brewer, 425 Oak Lane, Towson 4, Md. Filed July17, 1961, Ser. No. 124,575 12 Claims. (Cl. 195-439) This inventionrelates to an improved apparatus and method for culturingnucroorganismssucll as bacteria,

yeasts and molds.

My invention has particular application to an apparatus and method forautomatically inoculating successive units of culture media and forcontinuously culturing the microorganisms without the necessity forsuccessive manual inoculations.

The presently used [apparatus and methods for culturing microorganismssuch as bacteria, yeasts and molds present difficulties anddisadvantages. Thus, in order to maintain stock cultures it is necessaryto inoculate fresh culture media units at regular and repeated timeintervals. Failure to do this will result in the loss of certain stockcultures. Because of this, laboratories require the attendance ofskilled technicians to maintain the stock cultures during weekends andholiday periods if twenty four hour cultures are to be maintained.

In (addition, the apparatus heretofore used for culturing microorganismsof this type has been cumbersome, difiicul-t to handle and store and hasoccupied :a great deal of space. In addition, the inoculation requiresthe attention and attendance of highly skilled technicians, the use ofelaborate sterilization apparatus and elaborate care at each transfer ofthe culture in order to prevent contamination.

it is an object of the present invent-ion to overcome the diificultiesand disadvantages heretofore encountered and to provide an improvedmethod and apparatus in which the culturing process is a continuous onewith the inoculation of successive culture media units beingautomatically made.

A further object is the provision of a method and apparatus of the abovecharacter in which the apparatus is flexible and relatively compact andlight so that it is easy to handle and occupies a minimum amount ofspace; which is inexpensive and, therefore, is disposable; which doesnot require the close attention and attendance of a skilled technician;and which eliminates or minimizes the danger of contamination.

A further objectof the invention is the provision of an improved methodand apparatus of the above character which may be used in the generationand collecting of gas such as hydrogen, methane and carbon dioxide.

In carrying out my invention, I contemplate the pro vision of aplurality of separate units of culture media communicating with eachother only through interstices and pores of a size and varrangernent sothat the microorganisms to be cultured will slowly diffuse or growtherethrough whereby the units can be arranged in sequeuce and afterinoculation of the first unit of culture media the microorganisms willmultiply 'and will grow or diffuse successively from unit to unitthereby providing automatic inoculation and continuous culturing. Theapparatus and method can be controlled so that fixed predetermined timeintervals are measured by the inoculation and culturing of thesuccessive units. Thus, each unit mav represent a separate day of theyear so as to permit the study of the culturing of bacteria over anydesired calendar period. such as a year, without the necessity of manualinoculations.

My invention 13.1150 contemplates that by selection of the culturemedia, i.e. by utilizing carbohydrate media such as dextrose or glucose,sufficient hydrogen, methane 3,102,082 Patented Aug. 27, 1963 2 orcarbon dioxide gas can be generated on a program med schedule andcollected from the successive units.

In the accompanying drawings:

FIG. 1 is an elevational view of one form of apparatus embodying myinvention;

FIGS. 2 and 3 are sectional views in the direction of the arrows on thelines 22 and 33 of FIG. 1;

FIG. 4 is an elevational View similar to FIG. 1 of a modified apparatuswhich may be used in generating gas;

FIG. 5 is a fragmentary view of a further modified type of apparatus inthe form of a relatively elongated envelope; and

FIG. '6 is :a perspective view of the apparatus shown in FIG. 5 wound inhelical form and disposed in a cylind-rical container.

In the several forms of my invention I provide a casing which is inertand impermeable to the culture media and the microorganisms to becultured and I separate the interior of the casing by a plurality ofpartition means into a plurality of separate compartments each havingculture media therein. The partitions are provided with portions,preferably wicks extending through and sealed therein, havinginterstices and pores of a size and arrangement so that themicroorganisms to be cultured will slowly grow or diffuse therethrough.

In the 'form of my invention shown in FIGS. 1, 2 and 3, the casing is anenvelope 10 made of a flexible, plastic material inert to andimpermeable to the culture media and the microorganisms to be cultured.Suitable plastic materials for this purpose are polyethylene, polyvinylchloride and the co-polymers thereof, polypropylene, tetrailuoroethyleneand triiluorochloroethylene. Where the microorganisms or bacteria areanaerobic the plastic material should be impermeable to air. Where themicroorganisms or bacteria being cultured are aerobic, then the plasticmaterial should be permeable to air while at the same time beingimpermeable to the culture media.

Thin sheets of polyethylene and polypropylene in the order of .002"thick will transmit oxygen and are suitable for use with aerobicmicroorganisms. Thicker sheets of such material in the order of .006" ormore in thickness and also laminated thinner sheets generally do nottransmit oxygen. Where aerobic microorganisms are. to be cultured,either the entire envelope or only one side thereof may be made ofplastic material which transmits oxygen.

For certain purposes it is. preferred that the plastic material betransparent so that the bacteriologist or technician can observe themedia and culture inside the casing.

From the standpoint of ease and simplicity of manufacture I prefer toemploy a thermoplastic material so that heat sealing can be employed forsealing the casing and for forming the partitions therein.

The envelope illustrated in FIGS. 1 to 3 is formed of a single sheet ofplastic material folded upon itself along its lower edge 12 and thenheat sealed along the lateral edges 14 and across the top as at 16 so asto provide a completely sealed envelope. The top of the envelope asshown may be provided with apertures '18, preferably fitted withgrommets for supporting or suspending the envelope in vertical orupright position.

The interior of the envelope is provided with a plurality of verticalhorizontally spaced partitions formed by heat sealing the two plies ofthe envelope together from top to bottom as shown. The interior of theen velope is thereby divided into a plurality of successive compartments22. The partitions 20 are provided with portions having interstices orpores communicating with the culture media in the compartments on thetwo sides thereof and the interstices or pores should be of a size 3 andarrangement so that the microorganism being cultured will slowly grow ordiffuse therethrough.

For this purpose, I prefer to employ a Wick 24 extending through andsealed in each of the partitions 20 and immersed in the culture media ineach of the compartments. This can be simply accomplished by providing awick extending transversely from the first compartment through each ofthe compartments to the last compartment. The wick is sealed in place inthe partitions at the time that the heat sealed partitions 20 areformed.

The interstices or pores should be of a size so that when themicroorganisms multiply in one compartment they will grow or diffusethrough the wick to the next successive compartment. At the same timethe wick pore size should not be so great as to permit the free How ofthe culture media or of the metabolic products. The size of theinterstices or pores should be varied with the speed of the transferfrom compartment to compartment which is desired and also with themicroorganisms being cultured. For speedier transfer, larger pores or alarger crosssectional wick should be employed. The speed of the transfermay be decreased by using a wick with smaller pores or of smallercross-sectional area or by adding agar or gelatin to the culture media.

For this purpose, various types of filter media may be employed such asfilter paper or filter cloth, wet strength paper towelling, fibre glasstape, cotton ribbon, and cotton twill or cross grain ribbon. Commercialcotton twill tape .250 by .007 serves very satisfactorily.

Inside the envelope or casing in each of the compartments I place theculture media 2 6. Any of the well known culture media may be employedsuch as dilute solutions of peptones and dextrose, sucrose, glucose,maltose and other sugars, gelatin, agar-agar, serum, blood and the like.The culture meria is placed in the compartments before the final sealingof the upper end of the casing or envelope. As previously indicated, thespeed of the transfer of the microorganisms from compartment tocompartment can be in part controlled by the culture media. Byincreasing the amount of agar or gelatin, the speed of transfer can bedecreased.

After the envelope has been completed and the culture media inserted inthe compartments, the assembly is sterilized in suitable manner, in thecase of plastic materials which will Withstand high temperatures such aspolypropylene autoclaving at 15 pounds steam pressure at 121 C. for 20minutes or in the case of all of the plastic materials by radiation orby immersing the apparatus in an atmosphere of ethylene oxide.

in using my improved apparatus, I inoculate the first compartment asshown in FIG. 1, as for instance by inserting a hypodermic needletherein and injecting a small quantity of the microorganism or bacteriato be cultured. Upon withdrawal of the needle, the envelope or casingmay be rescaled with a strip of pressure sensitive tape. The envelope isthen maintained in the desired thermal environment by being suspended onhooks from the apertures 18 in an incubator or similar apparatus. Formost microorganisms, the temperature range will be between 20 and 45 C.In the case of psychrophiles the preferred temperature is between and 20C., in the case of mesophiles the preferred temperature is between 20and 45 C. and in the case of thermophiles the preferred temperature isbetween 45 and 70 C. While thus maintained in the desired thermalenvironment, the microorganisms will multiply in the first compartmentand will finally diffuse through the wick in the second compartmentwhere the microorganisms will further multiply and diffuse through thewick into the third compartment. This process of continuous culturingand automatic inoculating of successive compartments continuessequentially through the envelope to the last compartment.

Where a uniform wick having uniform interstices and pores throughout itslength is employed, the time sequence of progress from compartment tocompartment is uniform. By controlling the size and arrangement of theinterstices and pores and/ or the width of the wick and/ or thecharacter of the culture media, the time sequence can be at any desiredinterval. Thus, it can be arranged on an hourly or daily basis so thatsuccessive compartments are inoculated and cultured on successive hoursor days. By using all the compartments as shown in FIG. 1, a. period ofone week can be covered with separate cultures for each day. It will beappreciated that this can be accomplished on a monthly, yearly, or anyother desired time or calendar basis.

Where it is desired to carry on the continuous culturing over a longperiod of time, as for instance 365 successive days, the form of theenvelope can be changed to that shown in FIGS. 5 and 6. Thus, in FIGS. 5and 6 I have illustrated a relatively elongated envelope 30 made of asimilar plastic material as that described in connection with FIGS. 1 to3. The plastic sheet material is folded upon itself at 32 and heatsealed along its upper edge 36 and at its two ends. The elongated stripis divided by transverse partitions or heat seals 38 into separatecompartments 4t] and a wick 44 preferably in unitary form extendslongitudinally for the entire length of the envelope and extends througheach seal or partition and is sealed therein. Culture media is placed ineach compartment as in the first form of my invention and after sealingthe assembly is suitably sterilized.

The wick is immersed in the culture media 46 in each of the compartments40. The interstices and the pores of wick 44, the cross-sectional areaof the wick and the character of the culture media, are such that themicroorganisms in the envelope will grow or diffuse from one compartmentto another so as to culture the media therein on a daily basis. Anenvelope long enough to have 365 separate compartments is required tocover a yearly period. For convenience and compactness, this elongatedenvelope may be wound in helical form as shown in FIG. 6 and packed in acylindrical casing or cassette 50 during storage, shipment and use.

In using the apparatus shown in FIGS. 5 and 6 the media in the firstcompartment is inoculated in the manner shown in FIG. 1. The envelope inhelical form is packed in its casing and the assembly may be kept in thedesired thermal environment as previously indicated by resting on ashelf in an incubator. This permits the study of cultures as forinstance mutations of bacteria on a daily basis over an entire yearperiod.

In the apparatus shown in FIGS. 1 to 3 and 5 to 6 the compartments areshown as being of uniform size. It will be appreciated that thecompartments may be of varying size. Thus, the first compartment may berelatively larger to thereby develop starter cultures for use in variousfermentation processes and dairy products production.

My improved apparatus and method may also be used in the generation andcollection of various gases such as hydrogen, methane, CO and the likefor various purposes as for instance so as to permit the bacteriologistto check or confirm the nature of the microorganisms by an analysis ofthe gases.

In FIG. 4 I have shown apparatus suitable for this pur pose. Thisapparatus is generally the same as shown in FIGS. 1 to 3 with theexception that the heat sealed partitions 20' extend upwardly from thelower end of the casing to a point spaced from the upper end thereof sothat the compartments 22 intercommunicate at the upper end thereof. Inthis form of my apparatus I provide an outlet 60 adjacent the upper endof the casing above the partitions 20 so that the gas generated in eachcompartment as a result of the culturing may be collected through theoutlet. A suitable shut-off valve 62 may be provided for the outletasshown. Where my apparatus is used in generating gas I prefer to employ acarbohydrate culture media such as a dilute solution of glucose,dextrose, sucrose or other sugars and peptone, since this results in thegeneration of a greater quantity of gas.

In each form of my invention, suitable indicators may be added to any ofthese culture media, such as brom cresol purple, and after growth of theorganism a change in color would result, as well as turbidity, toindicate which compartment the organism had reached at any given time.

Specific examples of the culturing or microorganisms pursuant to myinvention are as follows:

Example I An envelope of the type shown in FIGS. 1 to 3 is made frompolypropylene plastic material (.006" th.), and has the followingdimensions: Unit length is 14 /2; unit width 7 /2"; compartment length13'; compartment width 2". Before final heat sealing, 20 ml. of culturemedium having the following composition per liter is inserted in eachcompartment: Trypticase-17 gms; phytone3 gms; NaClgms; K HPO 2.5 gms;and dextrose- 5 gms; pl-I7.2. The wick material employed was cottontwill tape .007 thick by /2" wide and it was in the form of a unitarywick extending transversely from the first compartment through each ofthe partitions to the last compartment. The bag is then sterilized by anappropriate method. The culture medium in the first compartment wasinoculated by a hypodermic needle with Clostridium welchii, an anaerobe,and the aperture left by the needle after inoculation was sealed with asuitable sealing tape having a pressure sensitive adhesive. The envelopewas suspended in an incubator in a thermal atmosphere of 35 C. Thebacteria in the first compartment multiply and after 24 hours diffuse orgrow through the wick and inoculate the medium in compartment #2 Wherethe bacteria again multiple and inoculate the medium in succeedingcompartments. It was observed that the bacteria proceeded sequentiallyin uniform time intervals from compartment to compartment and that theculture in each compartment was free from contamination.

Example 11 An envelope of the type shown in FIGS. 1 to- 3 is made fromvery thin polypropylene (.00'2") or polyethylene which will transmitoxygen and has the following dimensions: Unit width is 7 /2; unit length14 /2; compartment length 13''; compartment width 2". Before final heatsealing, 20 ml. of culture medium is inserted in each compartment. Theculture medium is whole cows milk. The wick material employed was cottontwill tape .007 thick x /2" wide and it was in the form of a unitarywick extending transversely from the first compartment through each ofthe partitions to the last compartment. The bag is then sterilized by anappropriate method. The culture medium in the first compartment wasinoculated by a hypodermic needle with Lactobacillus acidophilus, anaerobe, and the aperture left by the needle after inoculation was sealedwith a suitable sealing tape having a pressure sensitive adhesive. Theenvelope was suspended in an incubator in a thermal atmosphere of 45 C.The bacteria in the first compartment multiply and after 24 hoursdiffuse or grow through the wick and inoculate the medium in compartment#2. where the bacteria again multiply and inoculate the medium insucceeding compartments. It was observed that the bacteria proceededsequentially in uniform time intervals from compartment to compartmentand that the culture in each compartment was free from contamination.

Example 111 The elongated envelope similar to that shown in FIGS. 5 and6 is made from similar materials to that described in connection withExample '1. Each compartment has a dimension of approximately 1" wide x2" high. The

same twill tape is used as a wick. In this case, however, 36'5compartments are made and the whole is placed in a metal can orcassette. This particular arrangement is of value in carrying culturesfor a very long period of time, as for instance for a period of one yearwith transfers from compartment to compartment being made on a dailybasis. Suitable indicators may be added to any of these culture media,such as brom cresol purple, and after growth of the organism a change incolor would result, as well as turbidity, to indicate which compartmentthe organism had reached at any given time.

It will be appreciated that modifications may be made in theillustrative embodiments and examples of my invention within the scopeof the appended claims. the specific size, shape and configuration ofthe envelopes may be changed and the envelopes may he made of anysuitable materials having the indicated characteristics.

I claim:

1. Apparatus for use in culturing microorganisms comprising meansproviding a casing impermeable to culture media and the microorganismsto be cultured, partition means separating the interior of said casinginto separate culturing compartments, and culture media disposed insidesaid casing in said separate compartments, said partition meansincluding a portion having interstices and pores in communication withthe culture media in both compartments of a size and arrangement so thatthe microorganisms to be cultured will slowly grow and diffusetherethrough but so as to prevent the free flow of the culture mediatherethrough.

2. Apparatus for use in culturing microorganisms and for generating gascomprising means providing a casing impermeable to culture media and themicroorganisms to be cultured, partition means extending upwardly fromthe lower end of the casing and terminating short of the upper endthereof to divide the interior of the lower portion of the casing intoseparate culturing compartments which intercommunicate at the upperportion of the casing and culture media disposed in said casing in saidcompartments at a level lower than the upper end of the partition means,said partition means including a portion having intersttices and poresof a size and arrangement so that the microorganisms to be cultured willslowly grow and diffuse therethrough but so as to prevent the free flowof the culture media therethrough and an outlet for gas generated insaid compartments formed in said casing near the upper portion thereof.

3. Apparatus for use in culturing microorganisms comprising meansproviding a casing impermeable to bacteria and the microorganisms to becultured, partition means separating the interior of said casing intoseparate culturing compartments for holding culture media and wick meansextending through and sealed in said partion means so as to communicatewith both the adjacent compartments on opposite sides of the partitionmeans, said wick means having interstices and pores of a size andarrangement so that the microorganisms to be cultured will slowly growand diffuse therethrough but so as to prevent the free flow of theculture media therethrough.

4. Apparatus for use in culturing microorganisms comprising meansproviding a casing impermeable to culture media and the microorganismsto be cultured, partition means separating the interior of said casinginto separate culturing compartments, culture media disposed inside saidcasing in said compartments and wick means extending through and sealedin said partition means so as to communicate with the culture media inboth compartments on opposite sides of the partition means, said wickmeans having interstices and pores of a size and arrangement so that themicroorganisms to he cultured will slowly grow and difluse therethroughbut so as to prevent the free flow of the culture media therethrough.

5. Apparatus for use in culturing microorganisms and for generating gascomprising means providing a casing Thus,

impermeable to culture media and the microorganisms to be cultured,partition means extending upwardly from the lower end of the casing andterminating short of the upper end thereof so as to separate the lowerportion of the casing into separate compartments intercommunieating atthe upper end thereof, culture media disposed inside said casing in saidcompartments, and wick means extending through and sealed in saidpartition means and communieating with the culture media in thecompartments on both sides of said partition means, said wick meanshaving interstices and pores of a size and arrangement so that themicroorganisms to be cultured will slowly grow and diffuse therethroughbut so as to prevent the free flow of the culture media therethrough,and an outlet for the gas gen erated in said compartments formed in saidcasing at the upper portion thereof.

6. Flexible, disposable and relatively compact apparatus for use inculturing microorganisms comprising an en velope made of flexible,plastic material inert and impermeable to culture media and themicroorganisms to be cultured, partition means separating the interiorof said envelope into separate culturing compartments for holdingculture media and wick means extending through and sealed in saidpartition means and communicating with the compartments on both sidesthereof, said wick means having interstices and pores of a size andarrangement so that the microorganisms to be cultured will slowly growand diffuse therethrough but so as to prevent the free flow of theculture media therethrough.

7. Flexible, disposable and relatively compact apparatus for use inculturing aerobic microorganisms as set forth in claim 6 in which atleast a portion of the envelope is made of a plastic material which ispervious to oxygen.

8. Flexible, disposable and relatively compact apparatus for use inculturing microorganisms as set forth in claim '6, in which said wickmeans comprises cotton twill tape.

9. Flexible, disposable and relatively compact apparatus for use inculturing microorganisms and generating gas comprising an envelope madeof plastic material inert and impermeable to the culture media and themicroorganisms to be cultured, partition means extending upwardly fromthe lower end thereof and terminating short of the upper end forseparating the lower portion of the interior of the envelope intoseparate compartments, culture media disposed in said compartments at alevel lower than the upper ends of the partitions when the casing is inupright position, and wick means extending through and sealed in saidpartition means and communicating with the culture media in thecompartments on both sides thereof and an outlet for the gas generatedin said compartments formed in said casing above the upper end of saidpartitions, said wick means having interstices and pores of a size andarrangement so that the microorganisms to be cultured will slowly growand diffuse therethrough but so as to prevent the free flow of theculture media therethrough.

10. Apparatus for use in automatically inoculating and culturingmicroorganisms on a chronological basis comprising a relatively nalrowelongated envelope made of flexible, plastic material having a pluralityof spaced seals forming partitions extending across the envelope so asto divide the envelope into a plurality of separate culturingcompartments each representing a predetermined time interval withrespect to the adjacent compartment, culture media disposed inside saidenvelope in each of said compartments, and a continuous wick extendingthrough and sealed in each of said partitions and disposed in theculture media in each compartment, said wick having interstices andpores of a size and arrangement so that the microorganisms to becultured will slowly grow and diffuse through the portion sealed in eachpartition within the aforesaid predetermined time interval but so as toprevent the free flow of the culture media therethrough, said envelopebeing flexible and subject to being arranged in a tightly compactedhelix, and a circular container for holding said envelope when disposedin helical form.

11. The method for automatically and sequentially inoculating landculturing microorganisms in successive separate units of culture mediawhich comprises providing a plurality of separate culturing compartmentsin sequential arrangement with partitions beween the compartmentsprovided wih a section having interstices and pores of a size andarrangement so that the microorganisms to be cultured will slowly growand diffuse therethrough but so as to prevent the free flow of theculture media therethrough, inoculating the culture media in the firstcompartment with the microorganisms to be cultured and maintaining theentire assembly in a thermal environment to cause said microorganisms tomultiply and to diffuse sequentially through the partition sectionshaving interstices and pores from compartment to compartment.

'12. The method for automatically and sequentially inoculating andculturing microorganisms in successive separate units of culture mediawhich comprises providing an elongated flexible plastic envelope dividedinto a plurality of sequentially arranged compartments having culturemedia therein by partitions having wicks extending therethrough formedwith interstices and pores of a size and arrangement so that themicroorganisms to be cultured will slowly diffuse therethrough but so asto prevent the free flow of the culture media therethrough, inoculatingthe culture media in the first compartment with the microorganisms to becultured and maintaining the assembly in the proper thermal environmentto cause the microorganisms to multiply and diffuse sequentially throughthe wicks from compartment to compartment.

References Cited in the file of this patent UNITED STATES PATENTS2,954,327 Kantz Sept. 27, 1960 2,971,850 Barton Feb. 14, 1961 2,980,540T urpin Apr. 18, 1961

1. APPARATUS FOR USE IN CULTURING MICROORGANISMS COMPRISING MEANSPROVIDING A CASING IMPERMEABLE TO CULTURE MEDIA AND THE MICROORGANISMSTO BE CULTURED, PARTITION MEANS SEPARATING THE INTERIOR OF SAID CASINGINTO SEPARATE CULTURING COMPARTMENTS, AND CULTURE MEDIA DISPOSED INSIDESAID CASING IN SAID SEPARATE COMPARTMENTS, SAID PARTITION MEANSINCLUDING A PORTION HAVING INTERSTICES AND PORES IN COMMUNICATIN WITHTHE CULTURE MEDIA IN BOTH COMPARTMENTS OF A SIZE AND ARRANGEMENT SO THATTHE MICROORGANISMS TO BE CULTURED WILL SLOWLY GROW AND DIFFUSETHERETHROUGH BUT SO AS TO PREVENT THE FREE FLOW OF THE CULTURE MEDIATHERETHROUGH.